Team:Goettingen/week12-2

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                                <!-- Text body -->
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Language: <img height="20", src="http://www.patrickreinke.de/igem/eng.jpg"> English, <img height="20", src="http://www.patrickreinke.de/igem/deu.jpg"> <a href="https://2012.igem.org/Team:Goettingen/main_deu">Deutsch</a> <br>
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<h2><b>V07_09 </b></h2><br>
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<h2><b>V07_16 </b></h2><br>
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<b> Purification of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> </b><br>
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<b> Chemical retransformation of <i>fliC</i> (DH10B), <i>fliC</i> (Salmonella), <i>motA</i>, <i>motB</i> and <i>yhjH</i> into <i>E. coli</i> (DH10B, BL21, DH5&alpha; and XL1 Blue) </b><br>
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
The amplicons were separated via application on an 1% agarose gel and subsequently cut out. The DNA was purified out of the gel slices using peqGOLD Gel Extraction Kit (Peqlab) according to the user manual. <br></li>
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For the chemical retransformation the standard <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a> was followed. This time the four <i>E. coli</i> strains DH10B, BL21, DH5&alpha; and XL1 Blue were transformed in order to subsequently perform swimming assays and compare the accessibility of the different strains. <br></li>
 +
<li>Observations & Results: <br>
 +
The retransformation was succesful. Numerous colonies had formed at each plate except for the negative control.</li>
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<h2><b>V07_17 </b></h2><br>
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<h2><b>V07_10 </b></h2><br>
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<b> Preparation of over night cultures</b><br>
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<b>V07_10_1 Preparative double digestion of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i>, <i>yhjH</i> and pUC18 </b><br>
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<ul>
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<li>Experiment: <br>
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<li>Experiment: <br>
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In order to clone the new genes into pUC18, all components were digested with EcoRI and XbaI according to the protocol. The restricted plasmid pUC18 was subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. The other digestion products were investigated via preparation of an 1% analytical gel and directly purified using the peqGOLD Gel Extraction Kit (Peqlab) according to the manual as well. </li>
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In order to test the influence of the different genes on the motility of the distinct strains over night cultures were prepared. On that account it is important to shake the cultures only at 140 rpm to spare the flagella. <br></li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
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The digestion was successful. For all PCR products as well as for the vector band of the expected size could be obtained and the fragments can now be used for further cloning. </li>
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Not all cultures grew properly. Especially, those of <i>E.coli</i> BL21 featured a low cell density.</li>
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<br>
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<b>V07_10_2 Insertion of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> into pUC18</b><br>
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        <ul>
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                <li>Experiment:  <br>
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The ligation of the digested genes with the plasmid pUC18 was conducted as described in the protocol.
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</li>
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<h2><b>V07_11 </b></h2><br>
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<h2><b>V07_18 </b></h2><br>
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<b> Chemical transformation of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> into <i>E. coli</i> (DH10B)  </b><br>
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<b> Performance of motility assay</b><br>
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
For the chemical transformation the standard protocol was followed. <br></li>
+
LB medium containing ampicillin was inoculated with the over night culture and shaken until an optical density of 0.6 was reached. Meanwhile ampicillin containing M9 agar plates were prepared and autoclaved Whatman Filter Papers were imbued with tryptone solution. Then the cultures were dropped on the agar at a distance of 1 cm to the paper. On each plate also colonies hosting the empty vector pUC18 were dropped in order to have a reference. Each plate was prepared in duplicates.</li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
-
The transformation was successful since all plates showed numeours colonoes except the negative control. </li>
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The following freshly inoculated cultures did not grow very well and only reached an OD between 0.3 and 0.4:
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<i>fliC</i> (DH10B) in DH10B <br>
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<i>fliC</i> (DH10B) in DH5&alpha; <br>
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<i>fliC</i> (DH10B) in XL1 Blue <br>
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<i>motB</i> in DH5&alpha; <br>
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<h2><b>V07_12 </b></h2><br>
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<i>motB</i> in XL1 Blue <br>
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<b> Preparation of over night cultures</b><br>
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At the following day (19th of July) the colonies still looked exactly the same. Neither chemotaxis nor swimming could observed.
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<li>Experiment: <br>
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Two days after preparation of the swimming assay plates (20th of July) slight chemotaxis and motility could be perceived. <i>motB</i> and <i>fliC</i> overexpression seems to have a positive influence on bacterial motility in all strains.
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Over night cultures of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in pUC18 were prepared in order to isolate the plasmids. <br></li>
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At the 23rd of July quite big halos had formed. <i>motB</i> shows a strong effect, especially in DH10B. <i>fliC</i> (DH10B) seems to increase motility in all strains. However, the difference to the reference is not very drastic at all, but since the genes are not under control of strong promoters this results seems reasonable.  
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<h2><b>V07_13 </b></h2><br>
 
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<b>V07_13_1 Miniprep of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in pUC18</b><br>
 
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<li>Experiment: <br>
 
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Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.</li></ul>
 
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<b>V07_13_2 Test digestion of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in pUC18</b><br>
 
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<li>Experiment:  <br>
 
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All vectors were digested with EcoRI and XbaI according to the protocol. The restriction products were subsequently separated on a gel in order to determine successfully ligated plasmids.<br>
 
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<li>Observations & Results: <br>
 
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Except for <i>fliC</i> (Salmonella) #1 all picked colonies seem to host plasmids containing the aimed insert. </li>
 
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<b>Important pages</b>:<br>
 
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<a href="https://2012.igem.org/Team:Goettingen">Home</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Modeling">Modeling</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Notebook">Notebook</a>;
 
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Latest revision as of 17:18, 22 September 2012

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#2 Speed Improvement - 12th week

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V07_16


Chemical retransformation of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH into E. coli (DH10B, BL21, DH5α and XL1 Blue)
  • Experiment:
    For the chemical retransformation the standard protocol was followed. This time the four E. coli strains DH10B, BL21, DH5α and XL1 Blue were transformed in order to subsequently perform swimming assays and compare the accessibility of the different strains.
  • Observations & Results:
    The retransformation was succesful. Numerous colonies had formed at each plate except for the negative control.


V07_17


Preparation of over night cultures
  • Experiment:
    In order to test the influence of the different genes on the motility of the distinct strains over night cultures were prepared. On that account it is important to shake the cultures only at 140 rpm to spare the flagella.
  • Observations & Results:
    Not all cultures grew properly. Especially, those of E.coli BL21 featured a low cell density.


V07_18


Performance of motility assay
  • Experiment:
    LB medium containing ampicillin was inoculated with the over night culture and shaken until an optical density of 0.6 was reached. Meanwhile ampicillin containing M9 agar plates were prepared and autoclaved Whatman Filter Papers were imbued with tryptone solution. Then the cultures were dropped on the agar at a distance of 1 cm to the paper. On each plate also colonies hosting the empty vector pUC18 were dropped in order to have a reference. Each plate was prepared in duplicates.
  • Observations & Results:
    The following freshly inoculated cultures did not grow very well and only reached an OD between 0.3 and 0.4:
    fliC (DH10B) in DH10B
    fliC (DH10B) in DH5α
    fliC (DH10B) in XL1 Blue
    motB in DH5α
    motB in XL1 Blue

    At the following day (19th of July) the colonies still looked exactly the same. Neither chemotaxis nor swimming could observed. Two days after preparation of the swimming assay plates (20th of July) slight chemotaxis and motility could be perceived. motB and fliC overexpression seems to have a positive influence on bacterial motility in all strains. At the 23rd of July quite big halos had formed. motB shows a strong effect, especially in DH10B. fliC (DH10B) seems to increase motility in all strains. However, the difference to the reference is not very drastic at all, but since the genes are not under control of strong promoters this results seems reasonable.

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